ANTIMICROBIAL AGZNTS AND CHEMOTHERPY, May 1977, p. 791-796 Copyright 0 1977 American Society for Microbiology
Vol. 11, No. 5 Printed in U.S.A.
In Vitro Activity of Netilmicin Compared with Gentamicin, Tobramycin, Amikacin, and Kanamycin THEODORE C. EICKHOFF* AND JOSEPHINE M. EHRET Division of Infectious Disease, Department of Medicine, University of Colorado Medical Center, Denver, Colorado 80262 Received for publication 23 August 1976
The in vitro activity of netilmicin was compared with that of gentamicin, tobramycin, amikacin, and kanamycin against 636 strains of bacteria recently isolated from clinical sources. Gentamicin was the most active antibiotic, but netilmicin and tobramycin closely paralleled it. Netilmicin was generally fourto eightfold less active than gentamicin against Serratia and group A streptococci, and was twofold less active against Pseudomonas aeruginosa. When effects of inoculum size and concentration of divalent cations in the media were evaluated, netilmicin was shown to be similar to gentamicin in vitro. Minimum inhibitory concentrations for P. aeruginosa were increased as much as 18-fold when the Mg2+ and Ca2+ concentrations were increased to physiological levels in Mueller-Hinton broth. The increasing frequency of nosocomial infections caused by. gram-negative bacilli has become one of the major infectious disease problems of the 1970s (7). Escherichia, Klebsiella, Enterobacter, Pseudomonas, and Serratia nqw account for well over half of all hospital-acquired infections (5). The aminoglycosides are among the most useful drugs currently available for treating serious infections due to these genera, but the problems associated with their use, that is, development of resistant strains (10, 14, 15, 20) and potential toxicity (2, 3, 6, 9, 11, 12, 13, 19, 21), are well known. Because of its broad spectrum, gentamicin has been particularly useful in the treatment of such infections. One of the most worrisome factors in the use of gentamicin is the narrow margin between an effective dose and a toxic dose. Netilmicin, 0-2,6-diamino-2,3,4,6,-tetradeoxy-c-D-glycerol-hex-4-enopyranosyl-(1 -+ 4)0- [3-deoxy-4-C-methyl-3- (methylamino)-,3-L-
arabinopyranosyl-(1
-+
mirabilis, 46 Proteus rettgeri, 17 Serratia, 35 Salmonella, 10 Shigella, 75 Pseudomonas aeruginosa, 75 Staphylococcus aureus, 52 Staphylococcus epidermidis, 26 group D streptococci, 18 alpha-hemolytic streptococci (non-group D), 23 group A beta-hemolytic streptococci, 14 Streptococcus pneumoniae, 19 Neisseria gonorrhoeae, 18 Neisseria meningitidis, and 16 Haemophilus influenzae. Most of these organisms were recently isolated from clinical sources and identified in the Clinical Microbiology Laboratory of Colorado General Hospital under the direction of L. Barth Reller. The strains of N. gonorrhoeae were supplied by the Colorado General Hospital Venereal Disease Laboratory under the direction of Peter E. Dans. Most of the strains of Salmonella and Shigella were obtained from the Colorado State Public Health Laboratory. The P. rettgeri isolates were provided by F. Marc LaForce of the Denver Veterans Administration Hospital. Aminoglycosides. Standard laboratory reference powders of netilmicin and gentamicin sulfate were provided by the Schering Corp., amikacin and kanamycin sulfate were donated by Bristol Laboratories, and tobramycin standard solution was provided by Eli Lilly & Co. Susceptibility testing methods. (i) Broth minimal inhibitory concentration. The minimal inhibitory concentrations (MICs) of E. coli, Enterobacter, Proteus, Salmonella, Shigella, Klebsiella, Serratia, P. aeruginosa, S. aureus, S. epidermidis, group D streptococci, and S. pneumoniae were determined by a microtiter broth dilution technique. All tests were performed in standard Mueller-Hinton broth (pH 7.4, 7.2 mg of magnesium and 12 mg of calcium per liter), with the exception of group D streptococci and S. pneumoniae, which were tested in Trypticase soy broth (pH 7.35). The group D streptococci were also tested in Mueller-Hinton broth supplemented with 2% defibrinated sheep erythrocytes. All strains of
6)]-2-deoxy-1-N-ethyl-D-
streptamine, is a new semisynthetic aminoglycoside antibiotic that, based on subacute toxicity studies done in rats and dogs, may have less nephrotoxic and ototoxic potential than gentamicin (17). The purpose of this study is to compare the in vitro activity of netilmicin with that of gentamicin, tobramycin, amikacin, and kanamycin. MATERIALS AND METHODS Bacteria. A total of 636 strains of bacteria were tested. These were distributed as follows: 70 Escherichia coli, 35 Enterobacter, 52 Klebsiella, 35 Proteus 791
792
EICKHOFF AND EHRET
Pseudomonas were additionally tested in MuellerHinton broth (pH 7.38) adjusted to contain 31.2 mg of magnesium and 76.0 mg of calcium per liter. Magnesium and calcium levels in both the adjusted and unadjusted broth were determined in the Pediatric Microchemistry Laboratory of the University of Colorado Medical Center by means of atomic absorption spectrophotometry. Serial twofold dilutions of freshly prepared antibiotics were made in the appropriate broth, and 0.05 ml of each dilution was dropped into the wells of a microtiter plate (Cooke Engineering Co.). With the exception of S. pneumoniae, which was diluted 10-3, overnight broth cultures of the organisms were diluted 10-5 in the appropriate broth, and 0.05 ml was then added to the diluted antibiotic. The plates were placed on a shaker for 5 min, and then incubated overnight at 35°C in ambient air. The MIC was defined as the lowest concentration of antibiotic in which there was no visible growth. The minimal bactericidal concentration (MBC) was determined by using an adaptation of the Steers replicator (18), which delivered approximately 0.003 ml from each well of the microtiter plates to Mueller-Hinton agar. The strains of group D streptococci and S. pneumoniae were subcultured on Mueller-Hinton agar containing 4% defibrinated sheep erythrocytes. Subcultures of S. pneumoniae were incubated overnight at 35°C in a 5% CO2 atmosphere. All other subcultures were incubated overnight at 35°C in ambient air. (ii) Agar dilution method. The strains of N. gonorrhoeae, N. meningitidis, H. influenzae, group A beta-hemolytic streptococci and alpha-hemolytic streptococci (non-group D) were all tested by the twofold agar dilution method with the Steers inocula replicator (18). Neisseria and Haemophilus were inoculated onto plates containing GC medium base (Difco) supplemented with 1% hemoglobin (BBL) and 1% IsoVitaleX (BBL). Strains of the non-group D alpha-hemolytic streptococci and the group A beta-hemolytic streptococci were tested on Trypticase soy agar containing 4% defibrinated sheep erythrocytes. ESCHERICHIA COLI
ANTIMICROB. AGENTS CHZMOTHER. As inoculum, a volume of approximately 0.003 ml of an overnight undiluted broth culture was applied to the surface of the antibiotic-containing plates with the Steers replicator. Strains of Neisseria and Haemophilus were grown overnight in Mueller-Hinton broth supplemented with 1% hemoglobin and 1% IsoVitaleX at 35°C in a 5% CO2 atmosphere. For N. gonorrhoeae this provided an inoculum of approximately 1.5 x 108 colony-forming units (CFU) per ml; for N. meningitidis, 1.3 x 108 CFU/ml; and for H. influenzae, 1.5 x 108 CFU/ml. Group A beta-hemolytic streptococci and alphahemolytic streptococci were grown overnight in Trypticase soy broth supplemented with 2% defibrinated sheep erythrocytes. For the group A streptococci, this resulted in an inoculum of approximately 5 x 108 CFU/ml and, for the alpha-hemolytic streptococci, an inoculum of 2 x 107 CFU/ml. All of the plates were incubated at 35°C in a 5% CO2 atmosphere for 24 to 36 h. RESULTS
There were only rare instances of significant difference between MICs and MBCs; therefore, only MICs are shown. The activity of the five aminoglycosides against members of Enterobacteriaceae is shown in the cumulative distribution curves of Fig. 1 and 2. In general, there appears to be little significant in vitro difference among netilmicin, gentamicin, and tobramycin, except in the case of Serratia, for which gentamicin demonstrates. clearly greater activity. These aminoglycosides were consistently the most -active antibiotics against the Enterobacteriaceae, with amikacin the next most active and kanamycin the least active. The one striking exception to this was found when 46 strains of P. rettgeri were tested. Amikacin was fourfold more active than any of the other aminoglycosides tested, with 100% of strains in-
70 Strains
100' 90
a m
z
80* 70 60 50 40'
z
30'
-
20
I
10
x
Dl
02
04
08
KLEBSIELLA
u.
1.6
31
SPECIES
63 125
25
50
100
SERRATIA SPECIES
52 Strains
17 Strains
90
8~~~~~~~~~~~~0-
so-
>
70 X70 5- 60 l$?G6o. a 50-5o0 s40 4030 /..30 2020 to I10--
0.1
02
0.4 068
6
31
63
/
4/ ./
8
4 /
5O 100 02 04 08 ANTIBIOTIC CONCENTRATION mcg/mI lS2. 5
-
16
31
63
125
25
50
100
FIG. 1. Susceptibility of E. coli, Enterobacter, Klebsiella, and Serratia to netilmicin, gentamicin, tobramycin, amikacin, and kanamycin.
793
VOL. 11, 1977
IN VITRO ACTIVITY OF NETILMICIN
hibited at 6.3 ,ug/ml. This is particularly worthy of note, since disk susceptibility testing by the Kirby-Bauer method (1) had previously demonstrated that 55% of these strains were resistant to cephalothin, 73% to kanamycin, 100% to nitrofurantoin, 54% to ampicillin, 47% to gentamicin, and 43% to carbenicillin. Figure 3 demonstrates the activity of the aminoglycosides when tested against 75 strains of P. aeruginosa in standard Mueller-Hinton broth and in a calcium- and magnesium-adjusted Mueller-Hinton broth. These strains were divided among two groups; the first consisted of 31 strains reported as susceptible to
gentamicin when tested by the Kirby-Bauer method; the second consisted of 44 strains reported as intermediate or resistant by the Kirby-Bauer method. Tobramycin was the most active aminoglycoside against both groups of P. aeruginosa. Gentamicin, netilmicin, and amikacin were very similar in their activity. The Ca2+ and Mg2+ content of the broth was most significant when netilmicin and gentamicin were tested. In Mueller-Hinton broth unadjusted for divalent cation concentration, netilmicin had an activity against P. aeruginosa of approximately one dilution less than gentamicin, and was slightly more active than amika-
PROTEUS MIRASILIS
701
w
35 Strains
Q2
60-
4079
40A
20-
10-
cc
30
U 0
t1'
70
60
Z
46 Strins
PROTEUS RETTGERI
SA
02
01
20
30INE SeioeIGLtSPCEC 0She LA
04
SECE
08
SALMONELLA
5
3.1
1.6
n
SPECIES
35
10
6.3
12.5
25
50
100
0.2
0.1
04
SHIGELLA
treies
08
3i1
1.6
SPECIES
10
63
12'5
25
12-5
25
50
100
Stromn
100-
m
90-
90-
8070-
70-
60-
60-
50-
450-
w
40 3~~~~~~~~~0-
40-
30-
20-
2010
0.1
0.4
02
0.8
1.6
3.1
63
12.5
25
ANTIBIOTIC
50
1~00
0.
2
0.4
CONCENTRATION
0.B
1'6
6-3
FIG. 2. Susceptibility of P. mirabilis, P. rettgeri, Salmonella, and Shigella tobramycin, amikacin, and kanamycin.
PSEUDOMONAS AERUGINOSA 31 Streins Susceptible by the Kirby Bower Metlod Stendard Mueller Hinton Broth 90-
100 90 70 60
iI
Xo] iC
40 L-
,~
201
.
o
40 30 20
~
02 04 08 16 31 6.3 12.5 25 50 PSEUDOMONAS AERUGINOSA 44 Streins Intermeditc or Resistant by the Kirby Bower Method
Stenderd
Mueller
100
I
02
Hinton Broth 90
w
Ai I
4
10
01
cn
4O/
SO
0o
I-
gentamicin,
80
60
z
netilmicin,
PSEUDOMONAS AERUGINOSA 31 Stroins
so TO w
to
Susceptible by the Kirby Souer Method Co++ ond Mg++ Adjusted Mueller Hinton Broth
I/ /O.*
100
100
mcg/mI
04 08 1J6 3.1 63 125 25 50 100 PSEUDOMONAS AERUGINOSA 44 Strorns Intermedioet or Resistont by the Kirby Bouer Method Ce+ end M't+ Adjusted Mueller Hinton tBroth
I1,
w
Vol. 11, No. 5 Printed in U.S.A.
In Vitro Activity of Netilmicin Compared with Gentamicin, Tobramycin, Amikacin, and Kanamycin THEODORE C. EICKHOFF* AND JOSEPHINE M. EHRET Division of Infectious Disease, Department of Medicine, University of Colorado Medical Center, Denver, Colorado 80262 Received for publication 23 August 1976
The in vitro activity of netilmicin was compared with that of gentamicin, tobramycin, amikacin, and kanamycin against 636 strains of bacteria recently isolated from clinical sources. Gentamicin was the most active antibiotic, but netilmicin and tobramycin closely paralleled it. Netilmicin was generally fourto eightfold less active than gentamicin against Serratia and group A streptococci, and was twofold less active against Pseudomonas aeruginosa. When effects of inoculum size and concentration of divalent cations in the media were evaluated, netilmicin was shown to be similar to gentamicin in vitro. Minimum inhibitory concentrations for P. aeruginosa were increased as much as 18-fold when the Mg2+ and Ca2+ concentrations were increased to physiological levels in Mueller-Hinton broth. The increasing frequency of nosocomial infections caused by. gram-negative bacilli has become one of the major infectious disease problems of the 1970s (7). Escherichia, Klebsiella, Enterobacter, Pseudomonas, and Serratia nqw account for well over half of all hospital-acquired infections (5). The aminoglycosides are among the most useful drugs currently available for treating serious infections due to these genera, but the problems associated with their use, that is, development of resistant strains (10, 14, 15, 20) and potential toxicity (2, 3, 6, 9, 11, 12, 13, 19, 21), are well known. Because of its broad spectrum, gentamicin has been particularly useful in the treatment of such infections. One of the most worrisome factors in the use of gentamicin is the narrow margin between an effective dose and a toxic dose. Netilmicin, 0-2,6-diamino-2,3,4,6,-tetradeoxy-c-D-glycerol-hex-4-enopyranosyl-(1 -+ 4)0- [3-deoxy-4-C-methyl-3- (methylamino)-,3-L-
arabinopyranosyl-(1
-+
mirabilis, 46 Proteus rettgeri, 17 Serratia, 35 Salmonella, 10 Shigella, 75 Pseudomonas aeruginosa, 75 Staphylococcus aureus, 52 Staphylococcus epidermidis, 26 group D streptococci, 18 alpha-hemolytic streptococci (non-group D), 23 group A beta-hemolytic streptococci, 14 Streptococcus pneumoniae, 19 Neisseria gonorrhoeae, 18 Neisseria meningitidis, and 16 Haemophilus influenzae. Most of these organisms were recently isolated from clinical sources and identified in the Clinical Microbiology Laboratory of Colorado General Hospital under the direction of L. Barth Reller. The strains of N. gonorrhoeae were supplied by the Colorado General Hospital Venereal Disease Laboratory under the direction of Peter E. Dans. Most of the strains of Salmonella and Shigella were obtained from the Colorado State Public Health Laboratory. The P. rettgeri isolates were provided by F. Marc LaForce of the Denver Veterans Administration Hospital. Aminoglycosides. Standard laboratory reference powders of netilmicin and gentamicin sulfate were provided by the Schering Corp., amikacin and kanamycin sulfate were donated by Bristol Laboratories, and tobramycin standard solution was provided by Eli Lilly & Co. Susceptibility testing methods. (i) Broth minimal inhibitory concentration. The minimal inhibitory concentrations (MICs) of E. coli, Enterobacter, Proteus, Salmonella, Shigella, Klebsiella, Serratia, P. aeruginosa, S. aureus, S. epidermidis, group D streptococci, and S. pneumoniae were determined by a microtiter broth dilution technique. All tests were performed in standard Mueller-Hinton broth (pH 7.4, 7.2 mg of magnesium and 12 mg of calcium per liter), with the exception of group D streptococci and S. pneumoniae, which were tested in Trypticase soy broth (pH 7.35). The group D streptococci were also tested in Mueller-Hinton broth supplemented with 2% defibrinated sheep erythrocytes. All strains of
6)]-2-deoxy-1-N-ethyl-D-
streptamine, is a new semisynthetic aminoglycoside antibiotic that, based on subacute toxicity studies done in rats and dogs, may have less nephrotoxic and ototoxic potential than gentamicin (17). The purpose of this study is to compare the in vitro activity of netilmicin with that of gentamicin, tobramycin, amikacin, and kanamycin. MATERIALS AND METHODS Bacteria. A total of 636 strains of bacteria were tested. These were distributed as follows: 70 Escherichia coli, 35 Enterobacter, 52 Klebsiella, 35 Proteus 791
792
EICKHOFF AND EHRET
Pseudomonas were additionally tested in MuellerHinton broth (pH 7.38) adjusted to contain 31.2 mg of magnesium and 76.0 mg of calcium per liter. Magnesium and calcium levels in both the adjusted and unadjusted broth were determined in the Pediatric Microchemistry Laboratory of the University of Colorado Medical Center by means of atomic absorption spectrophotometry. Serial twofold dilutions of freshly prepared antibiotics were made in the appropriate broth, and 0.05 ml of each dilution was dropped into the wells of a microtiter plate (Cooke Engineering Co.). With the exception of S. pneumoniae, which was diluted 10-3, overnight broth cultures of the organisms were diluted 10-5 in the appropriate broth, and 0.05 ml was then added to the diluted antibiotic. The plates were placed on a shaker for 5 min, and then incubated overnight at 35°C in ambient air. The MIC was defined as the lowest concentration of antibiotic in which there was no visible growth. The minimal bactericidal concentration (MBC) was determined by using an adaptation of the Steers replicator (18), which delivered approximately 0.003 ml from each well of the microtiter plates to Mueller-Hinton agar. The strains of group D streptococci and S. pneumoniae were subcultured on Mueller-Hinton agar containing 4% defibrinated sheep erythrocytes. Subcultures of S. pneumoniae were incubated overnight at 35°C in a 5% CO2 atmosphere. All other subcultures were incubated overnight at 35°C in ambient air. (ii) Agar dilution method. The strains of N. gonorrhoeae, N. meningitidis, H. influenzae, group A beta-hemolytic streptococci and alpha-hemolytic streptococci (non-group D) were all tested by the twofold agar dilution method with the Steers inocula replicator (18). Neisseria and Haemophilus were inoculated onto plates containing GC medium base (Difco) supplemented with 1% hemoglobin (BBL) and 1% IsoVitaleX (BBL). Strains of the non-group D alpha-hemolytic streptococci and the group A beta-hemolytic streptococci were tested on Trypticase soy agar containing 4% defibrinated sheep erythrocytes. ESCHERICHIA COLI
ANTIMICROB. AGENTS CHZMOTHER. As inoculum, a volume of approximately 0.003 ml of an overnight undiluted broth culture was applied to the surface of the antibiotic-containing plates with the Steers replicator. Strains of Neisseria and Haemophilus were grown overnight in Mueller-Hinton broth supplemented with 1% hemoglobin and 1% IsoVitaleX at 35°C in a 5% CO2 atmosphere. For N. gonorrhoeae this provided an inoculum of approximately 1.5 x 108 colony-forming units (CFU) per ml; for N. meningitidis, 1.3 x 108 CFU/ml; and for H. influenzae, 1.5 x 108 CFU/ml. Group A beta-hemolytic streptococci and alphahemolytic streptococci were grown overnight in Trypticase soy broth supplemented with 2% defibrinated sheep erythrocytes. For the group A streptococci, this resulted in an inoculum of approximately 5 x 108 CFU/ml and, for the alpha-hemolytic streptococci, an inoculum of 2 x 107 CFU/ml. All of the plates were incubated at 35°C in a 5% CO2 atmosphere for 24 to 36 h. RESULTS
There were only rare instances of significant difference between MICs and MBCs; therefore, only MICs are shown. The activity of the five aminoglycosides against members of Enterobacteriaceae is shown in the cumulative distribution curves of Fig. 1 and 2. In general, there appears to be little significant in vitro difference among netilmicin, gentamicin, and tobramycin, except in the case of Serratia, for which gentamicin demonstrates. clearly greater activity. These aminoglycosides were consistently the most -active antibiotics against the Enterobacteriaceae, with amikacin the next most active and kanamycin the least active. The one striking exception to this was found when 46 strains of P. rettgeri were tested. Amikacin was fourfold more active than any of the other aminoglycosides tested, with 100% of strains in-
70 Strains
100' 90
a m
z
80* 70 60 50 40'
z
30'
-
20
I
10
x
Dl
02
04
08
KLEBSIELLA
u.
1.6
31
SPECIES
63 125
25
50
100
SERRATIA SPECIES
52 Strains
17 Strains
90
8~~~~~~~~~~~~0-
so-
>
70 X70 5- 60 l$?G6o. a 50-5o0 s40 4030 /..30 2020 to I10--
0.1
02
0.4 068
6
31
63
/
4/ ./
8
4 /
5O 100 02 04 08 ANTIBIOTIC CONCENTRATION mcg/mI lS2. 5
-
16
31
63
125
25
50
100
FIG. 1. Susceptibility of E. coli, Enterobacter, Klebsiella, and Serratia to netilmicin, gentamicin, tobramycin, amikacin, and kanamycin.
793
VOL. 11, 1977
IN VITRO ACTIVITY OF NETILMICIN
hibited at 6.3 ,ug/ml. This is particularly worthy of note, since disk susceptibility testing by the Kirby-Bauer method (1) had previously demonstrated that 55% of these strains were resistant to cephalothin, 73% to kanamycin, 100% to nitrofurantoin, 54% to ampicillin, 47% to gentamicin, and 43% to carbenicillin. Figure 3 demonstrates the activity of the aminoglycosides when tested against 75 strains of P. aeruginosa in standard Mueller-Hinton broth and in a calcium- and magnesium-adjusted Mueller-Hinton broth. These strains were divided among two groups; the first consisted of 31 strains reported as susceptible to
gentamicin when tested by the Kirby-Bauer method; the second consisted of 44 strains reported as intermediate or resistant by the Kirby-Bauer method. Tobramycin was the most active aminoglycoside against both groups of P. aeruginosa. Gentamicin, netilmicin, and amikacin were very similar in their activity. The Ca2+ and Mg2+ content of the broth was most significant when netilmicin and gentamicin were tested. In Mueller-Hinton broth unadjusted for divalent cation concentration, netilmicin had an activity against P. aeruginosa of approximately one dilution less than gentamicin, and was slightly more active than amika-
PROTEUS MIRASILIS
701
w
35 Strains
Q2
60-
4079
40A
20-
10-
cc
30
U 0
t1'
70
60
Z
46 Strins
PROTEUS RETTGERI
SA
02
01
20
30INE SeioeIGLtSPCEC 0She LA
04
SECE
08
SALMONELLA
5
3.1
1.6
n
SPECIES
35
10
6.3
12.5
25
50
100
0.2
0.1
04
SHIGELLA
treies
08
3i1
1.6
SPECIES
10
63
12'5
25
12-5
25
50
100
Stromn
100-
m
90-
90-
8070-
70-
60-
60-
50-
450-
w
40 3~~~~~~~~~0-
40-
30-
20-
2010
0.1
0.4
02
0.8
1.6
3.1
63
12.5
25
ANTIBIOTIC
50
1~00
0.
2
0.4
CONCENTRATION
0.B
1'6
6-3
FIG. 2. Susceptibility of P. mirabilis, P. rettgeri, Salmonella, and Shigella tobramycin, amikacin, and kanamycin.
PSEUDOMONAS AERUGINOSA 31 Streins Susceptible by the Kirby Bower Metlod Stendard Mueller Hinton Broth 90-
100 90 70 60
iI
Xo] iC
40 L-
,~
201
.
o
40 30 20
~
02 04 08 16 31 6.3 12.5 25 50 PSEUDOMONAS AERUGINOSA 44 Streins Intermeditc or Resistant by the Kirby Bower Method
Stenderd
Mueller
100
I
02
Hinton Broth 90
w
Ai I
4
10
01
cn
4O/
SO
0o
I-
gentamicin,
80
60
z
netilmicin,
PSEUDOMONAS AERUGINOSA 31 Stroins
so TO w
to
Susceptible by the Kirby Souer Method Co++ ond Mg++ Adjusted Mueller Hinton Broth
I/ /O.*
100
100
mcg/mI
04 08 1J6 3.1 63 125 25 50 100 PSEUDOMONAS AERUGINOSA 44 Strorns Intermedioet or Resistont by the Kirby Bouer Method Ce+ end M't+ Adjusted Mueller Hinton tBroth
I1,
w
